Oxidative stress and accelerated vascular aging: implications for cigarette smoking

Abstract

Cigarette smoking is the major cause of preventable morbidity and mortality in the United States and constitutes a major risk factor for atherosclerotic vascular disease, including coronary artery disease and stroke. Increasing evidence supports the hypothesis that oxidative stress and inflammation provide the pathophysiological link between cigarette smoking and CAD. Previous studies have shown that cigarette smoke activates leukocytes to release reactive oxygen and nitrogen species (ROS/RNS) and secrete pro-inflammatory cytokines, increases the adherence of monocytes to the endothelium and elicits airway inflammation. Here we present an overview of the direct effects of water-soluble cigarette smoke constituents on endothelial function, vascular ROS production and inflammatory gene expression. The potential pathogenetic role of peroxynitrite formation, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation in cardiovascular complications in smokers are also discussed.

Figure 1

Representative confocal images of nuclear ethidium bromide (EB) staining of en face preparations of rat aortas incubated with cigarette smoke extract (CSE; 4 μg/mL, for 6 h; Panel B) and untreated controls (Panel A). Vessels were incubated with the dye dihydroethidium, which produces a red nuclear fluorescence when oxidized to EB by O₂^.-. On these images the EB-stained elongated nuclei of vascular smooth muscle cells and the round nuclei of endothelial cells are visualized. Identical results were observed in 4 separate experiments (original magnification: 20X).

Figure 2

Incubation with cigarette smoke extract (CSE) elicits mitochondrial oxidative stress. Representative fluorescent images show intensive MitoSox staining (red fluorescence) in CSE-treated cells (CSE; 4 μg/mL, for 6 h; Panel B), whereas MitoSox fluorescence is significantly weaker in mitochondria of untreated control cells (Panel A). Cells were incubated with the mitochondrion-targeted O₂^.- sensitive dye MitoSox (Invitrogen), which produces a red fluorescence in the mitochondria when oxidized by O₂^.-. The DNA-binding dye Hoechst 33258 was used for nuclear counterstaining (original magnification: 20X).

Figure 3

Water-soluble cigarette smoke constituents elicit endothelial DNA damage. Coronary arterial endothelial cells were treated with cigarette smoke extract (CSE for 6 h). Then, cells were harvested and the extent of DNA damage was examined by single-cell electrophoresis (“comet assay”) as we reported (109, 139). Damaged DNA migrates during electrophoresis from the nucleus towards the anode, forming a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (relaxed and broken DNA). Frequency distribution of tail DNA content in untreated control and CSE-exposed endothelial cells were obtained (median values for tail DNA content are shown in the graph).

Figure 4

Water-soluble cigarette smoke constituents up-regulate vascular inflammatory cytokine expression. Isolated rat carotid arteries were maintained in organoid culture in the presence and absence of cigarette smoke extract (CSE) and mRNA expression of TNFα (A), IL-6 (B) and IL-1β (C) were assessed by real-time QRT-PCR, as reported (48). β-actin was used for internal normalizations. Some vessels were pre-incubated with the potent PARP inhibitor PJ34 (142-144, 146). Data are mean ± S.E.M. (n=4-6 for each group). *P<0.05 vs. untreated, # P<0.05 vs. CSE only.

Figure 5

Water-soluble cigarette smoke constituents elicit NF-κB activation and enhance monocyte adhesiveness via increasing NAD (P)H oxidase-derived ROS generation in primary coronary arterial endothelial cells (CAECs). Panel A: Reporter gene assay showing the effect of cigarette smoke extract (CSE) on NF-κB reporter activity in CAECs. The effects of pre-treatment with NAD (P)H oxidase inhibitor apocynin (3×10^-4 mol/L) on CSE-induced NF-κB reporter activity in CAECs is also shown. Endothelial cells were transiently co-transfected with NF-κB-driven firefly luciferase and CMV-driven ranilla luciferase constructs followed by CSE stimulation. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was obtained from four to seven independent transfections. *p<0.05. (Data are mean ± S.E.M. *p<0.05. vs. untreated; #P<0.05 vs. CSE only). B: Treatment of carotid arteries and aortas with cigarette smoke extract (CSE) significantly increased adhesion of fluorescently labeled PMA-stimulated monocyte enriched peripheral blood mononuclear cells (PBMC). The effects of CSE were also assessed after pretreatment with the NAD (P)H oxidase inhibitor apocynin (3×10^-4 mol/L) and DPI (10^-5 mol/L) or catalase (200 U/mL). Data are mean ± S.E.M. *p<0.05. vs. control. C: Treatment of CAECs with CSE significantly increased the adhesion of fluorescently labeled PMA-stimulated monocytes. Pre-treatment with the NAD (P)H oxidase inhibitor apocynin significantly attenuated CSE-induced monocyte adhesion. Data are mean ± S.E.M. *p<0.05. vs. control. #p<0.05 vs. CSE alone. Figure is redrawn based on data from reference (48).

Figure 6

Water-soluble cigarette smoke constituents elicit PARP activation in human coronary arterial endothelial cells. Endothelial cells were treated with cigarette smoke extract (CSE). Then, cells were harvested and the poly (ADP-ribose) polymer content was assessed by Western blotting.

Figure 7

Proposed scheme for the mechanisms by which water soluble components of cigarette smoke promote pro-inflammatory phenotypic alterations in the blood vessels. The model predicts that cigarette smoke induces an increased generation O₂^.- by NAD (P)H oxidase, which scavenges vasodilator NO resulting in an enhanced ONOO- formation and endothelial dysfunction. Cigarette smoke also impairs mitochondrial function eliciting mitochondrial oxidative stress. Increased cellular levels of NAD (P)H oxidase- and mitochondria-derived O₂^.-, H₂O₂ and/or ONOO- activate redox-sensitive signaling pathways, including MAP kinases and the transcription factor NF-κB, up-regulating inflammatory gene expression. Oxidative-nitrosative stress (in particular, the enhanced ONOO- levels and increased production of hydroxyl radicals) also elicits nuclear DNA damage promoting endothelial apoptosis and activating the nuclear enzyme PARP-1, which importantly contributes to NF-κB -dependent regulation of gene transcription. The resulting pro-inflammatory vascular phenotype will likely increase monocyte recruitment to the vascular wall and promote the development of atherosclerosis, especially if other risk factors (e.g. hypertension, hypercholesterolemia, hyperhomocysteinemia) are also present.